Anti-c-Met/anti-EGFR bispecific antibodies

ABSTRACT

There are provided an anti-c-Met/anti-EGFR bispecific antibody, a method for treating cancer using the same, and an anti-EGFR scFv fragment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2013-0034890 filed on Mar. 29, 2013, in the Korean Intellectual Property Office, the entire disclosures of which are herein incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 138,777 Byte ASCII (Text) file named “715753SequenceListing_revised_20160614. TXT” created on Jun. 14, 2016.

BACKGROUND OF THE INVENTION

1. Field

Provided are an anti-c-Met/anti-EGFR bispecific antibody, a method of preventing and/or treating a cancer using the same, and an anti-EGFR scFV fragment.

2. Description of the Related Art

c-Met and EGFR (or HER family) interact with each other and are involved in various mechanisms related to tumors. These proteins (targets) are typical receptor tyrosine kinases (RTKs) present at the surface of cells, thereby inducing the proliferation of cancer cells, the penetration of the cancer cells, angiogenesis, etc. Also, these proteins participate in each other's signal transduction systems by interacting with each other, thereby inducing resistance against each other's therapeutic agents.

Multispecific antibodies targeting two or more antigens have been developed in various kinds and forms and are expected as a new drug antibody having excellent therapeutic effects compared to a monoclonal antibody. Most of multispecific antibodies have been developed so that their therapeutic effects on cancers can be increased by recognizing an antigen of cytotoxic cells (killer cells) and an antigen of cancer cells at the same time, wherein the cancer cells are killed by the cytotoxic cells. However, when considering that the research results reveal that cancer cells themselves can be mutated to proliferate and penetrate even by intracellular ligands or various antigens of the same cancer cells other than the targeted antigen, it is expected that a multispecific antibody capable of recognizing an antigen of the cancer cells as well as an antigen of the killer cells will be also useful in treating cancers.

Accordingly, there is a desire for the development of a multispecific antibody to achieve effective cancer treatment effects by recognizing two or more kinds of antigens in cancer cells at the same time.

BRIEF SUMMARY OF THE INVENTION

One embodiment provides an anti-c-Met/anti-EGFR bispecific antibody comprising (a) an anti-c-Met antibody or an antigen-binding fragment thereof and (b) an anti-EGFR antibody or an antigen-binding fragment thereof, wherein the anti-c-Met antibody or the antigen-binding fragment thereof specifically binds to an epitope comprising 5 or more contiguous amino acids within the SEMA domain of c-Met protein, and the SEMA domain comprises the amino acid sequence of SEQ ID NO: 79.

Another embodiment provides a pharmaceutical composition for preventing and/or treating a cancer including the anti-c-Met/anti-EGFR bispecific antibody as an active ingredient.

Another embodiment provides a method of prevention and/or treatment of a cancer, including administering a pharmaceutically effective amount of the anti-c-Met/anti-EGFR bispecific antibody to a patient in need of the prevention and treatment of the cancer.

Another embodiment provides a use of the anti-c-Met/anti-EGFR bispecific antibody for the prevention and/or treatment of a cancer.

Still another embodiment provides an anti-EGFR scFv fragment including a heavy chain variable region of the anti-EGFR antibody, a light chain variable region of the anti-EGFR antibody, or a combination of the heavy chain variable region of the anti-EGFR antibody and the light chain variable region of the anti-EGFR antibody. In particular, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 114 is provided. A heavy chain variable region of an anti-EGFR antibody comprising the amino acid sequence of SEQ ID NO: 113 is provided. A light chain variable region of an anti-EGFR antibody comprising the amino acid sequence of SEQ ID NO: 114 is provided. An anti-EGFR scFv fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 113 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 114 is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 is a schematic diagram showing the structure of an anti-c-Met/anti-EGFR bispecific antibody according to one example.

FIG. 2 is a graph showing dual binding of an anti-c-Met/anti-EGFR bispecific antibody according to one example.

FIG. 3 contains graphs showing proliferation degrees of cancer cells when treated with an anti-c-Met/anti-EGFR bispecific antibody according to one example as relative values against the control group (M; antibody non-treatment group). The top graph is an MKN45 stomach cancer cell line, the middle graph is an EBC-1 lung cancer cell line, and the bottom graph is HCC827 ER#15, which is a stomach cancer cell line having resistance to Erlotinib (Er).

FIG. 4 illustrates western blotting results showing c-Met and EGFR activation inhibitory degrees of an anti-c-Met/anti-EGFR bispecific antibody according to one example in a MKN45 stomach cancer cell line.

FIG. 5 illustrates western blotting results showing c-Met and EGFR activation inhibitory degrees of an anti-c-Met/anti-EGFR bispecific antibody according to one example in an HCC827 WT cell line and total amounts of c-Met and EGFR.

FIG. 6 illustrates western blotting results showing c-Met and EGFR activation inhibitory degrees of an anti-c-Met/anti-EGFR bispecific antibody according to one example in HCC827 resistance microbes (HCC827 #15) and total amounts of c-Met and EGFR.

FIG. 7 contains confocal microscope images showing the co-localization of c-Met and EGFR by an anti-c-Met/anti-EGFR bispecific antibody according to one example in a MKN45 stomach cancer cell line.

FIG. 8 contains confocal microscope images showing the co-localization of c-Met and EGFR by an anti-c-Met/anti-EGFR bispecific antibody according to one example in an EBC-1 lung cancer cell line.

FIG. 9 is a graph showing binding activity of an anti-c-Met/anti-EGFR bispecific antibody according to one example to EGFR, which shows the stability of the bispecific antibody.

DETAILED DESCRIPTION OF THE INVENTION

The pre-existing targeting drugs which recognize only EGFR, a typical target widely expressed on cancer cells, induce over-expression and mutation of c-Met, allowing cancer cells to acquire resistance against the drugs, whereby the therapeutic effects of the drugs could be reduced. In the present invention, it is verified that a bispecific antibody recognizing c-Met and EGFR at the same time prevents the development of resistance and shows excellent cancer cell inhibitory effects, even in cancer cells having resistance, by previously blocking c-Met-implicated signal transduction which causes resistance against drugs.

Various bispecific antibodies have been developed, but their efficiency was not proved in clinical tests or several side effects were observed. For these reasons, there were many cases which were not approved by FDA and were not marketed as therapeutic antibodies. In spite of the fact that bispecific antibodies having various forms and mechanisms have been developed, the bispecific antibodies were not marketed due to a problem in the stability and productivity of the antibodies. In the production of early bispecific antibodies having an IgG form, it was very difficult to separate and purify a desired kind of bispecific antibodies due to random combination between light chains and heavy chains of antibodies. This becomes an obstacle in the mass production of the bispecific antibodies. Also, in case of bispecific antibodies with other than IgG forms, their stabilities as a drug were not verified in fields such as protein folding, pharmacokinetics, and the like.

In the present invention, it is verified that a bispecific antibody in which an anti-c-Met antibody is fused to an antibody recognizing a secondary target, EGFR, or an antigen binding fragment thereof (e.g., scFv) could improve the stability issue which was the biggest problem of the pre-existing bispecific antibodies.

One embodiment provides an anti-c-Met/anti-EGFR bispecific antibody including an anti-c-Met antibody or an antigen binding fragment thereof, and an anti-EGFR antibody or an antigen binding fragment thereof.

The antigen binding fragment may be selected from the group consisting of scFv, (scFv)2, Fab, Fab′, and F(ab′)2.

The “c-Met protein” refers to a receptor tyrosine kinase binding to hepatocyte growth factor. The c-Met proteins may be derived from any species, for example, those derived from primates such as human c-Met (e.g., GenBank Accession No. NP_000236) and monkey c-Met (e.g., Macaca mulatta, GenBank Accession No. NP_001162100), or those derived from rodents such as mouse c-Met (e.g., GenBank Accession No. NP_032617.2) and rat c-Met (e.g., GenBank Accession No. NP_113705.1). The proteins include, for example, a polypeptide encoded by the nucleotide sequence deposited under GenBank Accession No. NM_000245, or a protein encoded by the polypeptide sequence deposited under GenBank Accession No. NM_000236, or extracellular domains thereof. The receptor tyrosine kinase c-Met is involved in several mechanisms including cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.

The “EGFR (epidermal growth factor receptor)” is a member of the receptor tyrosine kinases (RTKs) of HER family. The binding of a ligand to the extracellular domain of EGFR induces receptor homo- or hetero dimerization with other ErbB receptors, which in turn results in intracellular self-phosphorylation of specific tyrosine residues. EGFR self-phosphorylation leads to downstream signal transduction networks including MAPK and PI3K/Akt activation which affects cell proliferation, angiogenesis and metastasis. Over-expression, gene amplification, mutation, or rearrangement of EGFR are frequently observed in several human malignant tumors and are related to poor prognosis of cancer treatment and bad clinical outcomes. For such reasons, EGFR becomes an important target in anticancer therapy. EGFR or HER2 may be derived from mammals, for example, primates such as humans and monkeys, or rodents such as rats and mice. For instance, the EGFR may be polypeptides encoded by the nucleotide sequences (mRNA) deposited under GenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, or NM_201283.1.

In one embodiment, the anti-c-Met/anti-EGFR bispecific antibody may comprise an anti-c-Met antibody or an antigen binding fragment thereof, and an anti-EGFR antibody or an antigen binding fragment thereof which is linked to the C terminus or N terminus, for example, C terminus, of the anti-c-Met antibody or the antigen binding fragment thereof.

In the anti-c-Met/anti-EGFR bispecific antibody, in order to fully perform the anti-c-Met antibody's activity to mediate intracellular migration and degradation of c-Met proteins, it may be advantageous that the anti-c-Met antibody has its own intact antibody structure. In addition, in case of the anti-EGFR antibody, its specific recognition and binding to EGFR is important, and thus it will be fine that just an antigen-binding fragment recognizing EGFR is included in the bispecific antibody. Therefore, the anti-c-Met/anti-EGFR bispecific antibody may comprise a complete anti-c-Met antibody (e.g., IgG type antibody) and an antigen binding fragment of the anti-EGFR antibody linked to the C terminus of the anti-c-Met antibody.

In the anti-c-Met/anti-EGFR bispecific antibody, the anti-c-Met antibody or the antigen binding fragment thereof, and the anti-EGFR antibody or the antigen binding fragment thereof, may be linked via a peptide linker or without it. Furthermore, a heavy chain portion and a light chain portion within the antigen binding fragment, for example, a heavy chain variable region and a light chain variable region within the scFv fragment, may be linked via a peptide linker or without it. The peptide linker which links the anti-c-Met antibody or the antigen binding fragment thereof, and the anti-EGFR antibody or the antigen binding fragment thereof, and the peptide linker which links the heavy chain portion and the light chain portion within the antigen binding fragment may be identical or different. The peptide linker may be 1 to 100, particularly 2 to 50, amion acids in length and include any kinds of amino acids. The peptide linker may include for example, Gly, Asn and/or Ser residues, and also include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for the peptide linker are known in the pertinent art. The length of the peptide linker may be determined within such a limit that the functions of the fusion protein will not be affected. For instance, the peptide linker may be formed by including a total of 1 to 100, 2 to 50, or 5 to 25 of one or more amino acids selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented as (G₄S)_(n), wherein n is a repeat number of (G₄S), which is an integer of 1 to 10, particularly an integer of 2 to 5.

In a particular embodiment, the anti-EGFR antibody may be selected from the group consisting of cetuximab (Erbitux), panitumumab, an anti-EGFR antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 109 and the light chain variable region of the amino acid sequence of SEQ ID NO: 111, and anti-EGFR antibody including the heavy chain variable region of the amino acid sequence of SEQ ID NO: 113 and the light chain variable region of the amino acid sequence of SEQ ID NO: 114.

The “antigen binding fragment” refers to a fragment of a full immunoglobulin structure including parts of the polypeptide including a portion of antigen-binding regions capable of binding to an antigen. For example, the antigen binding fragment may be scFv, (scFv)₂, Fab, Fab′, or F(ab′)₂, but is not be limited thereto.

Of the antigen binding fragments, Fab is a structure having variable regions of a light chain and a heavy chain, a constant region of the light chain, and the first constant region (C_(H1)) of the heavy chain, and it has one antigen binding site.

Fab′ is different from Fab in that it has a hinge region including one or more cysteine residues at the C-terminal of heavy chain C_(H1) domain. An F(ab′)₂ antibody is formed through disulfide bond of the cysteine residues at the hinge region of Fab′.

Fv is a minimal antibody piece having only a heavy chain variable region and light chain variable region, and a recombinant technique for producing the Fv fragment is well known in the pertinent art. Two-chain Fv may have a structure in which the heavy chain variable region is linked to the light chain variable region by a non-covalent bond, and single-chain Fv (scFv) may generally have a dimer structure as in the two-chain Fv in which the variable region of a heavy chain and the variable region of a light chain are covalently linked via a peptide linker or they are directly linked to each other at the C-terminal thereof. The peptide linker may be the same as described in the above, for example, a peptide linker having a length of 1 to 100, 2 to 50, particularly 5 to 25, amino acids, wherein any kinds of amino acids may be included without any restrictions.

In the anti-EGFR antibody or an antigen-binding fragment thereof, the portion of the light chain and the heavy chain excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, that is the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

The antigen binding fragments may be obtained using proteases (for example, a whole antibody is digested with papain to obtain Fab fragments, and is digested with pepsin to obtain F(ab′)₂ fragments), and may be prepared by a genetic recombinant technique.

In a particular embodiment, the anti-c-Met/anti-EGFR bispecific antibody may comprise an anti-c-Met antibody, and an scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂, for example, scFv, of an anti-EGFR antibody linked to the C terminus of the anti-c-Met antibody. For instance, scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂ of the anti-EGFR antibody may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 113, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 114.

Hence, in a particular embodiment, the anti-c-Met/anti-EGFR bispecific antibody may comprise an anti-c-Met antibody, and an scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂ of an anti-EGFR antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 113, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 114, linked to the C terminal of the anti-c-Met antibody.

In another particular embodiment, the anti-c-Met/anti-EGFR bispecific antibody may comprise an anti-c-Met antibody and an antigen binding fragment (scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂) of Cetuximab or an antigen binding fragment (scFv, (scFv)₂, Fab, Fab′ or F(ab′)₂) of Panitumumab, linked to the C terminal of the anti-c-Met antibody.

The anti c-Met antibody may be any one recognizing a specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope. It may be any antibody or antigen-binding fragment that acts on c-Met to induce intracellular internalization and degradation of c-Met.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may comprise the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region comprising the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. The region acts as an epitope for the specific anti-c-Met antibody of the present invention.

The term “epitope” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including 5 or more contiguous (consecutive or non-consecutive) amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide including 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide comprises the amino sequence of SEQ ID NO: 73 (EEPSQ), which serves as an essential element for the epitope. For example, the epitope may be a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope comprising the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope comprising the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which includes 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody may be an antibody or an antigen-binding fragment thereof, which comprises:

(i) at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence comprising 8-19 consecutive amino acids within the amino acid sequence of SEQ ID NO: 2 comprising amino acid residues from the 3^(rd) to 10^(th) positions of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 85, or an amino acid sequence having 6-13 consecutive amino acids within the amino acid sequence of SEQ ID NO: 85 comprising amino acid residues from the 1^(st) to 6^(th) positions of the amino acid sequence of SEQ ID NO: 85, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;

(ii) at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 86, or an amino acid sequence comprising 9-17 consecutive amino acids within the amino acid sequence of SEQ ID NO: 89 comprising amino acid residues from the 1^(st) to 9^(th) positions of the amino acid sequence of SEQ ID NO: 89, or a light chain variable region comprising the at least one light chain complementarity determining region;

(iii) a combination of the at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

(iv) a combination of the heavy chain variable region and the light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:

Formula I: Xaa₁-Xaa₂-Tyr-Tyr-Met-Ser (SEQ ID NO: 4), wherein Xaa₁ is absent or Pro or Ser, and Xaa₂ is Glu or Asp,

Formula II: Arg-Asn-Xaa₃-Xaa₄-Asn-Gly-Xaa₅-Thr (SEQ ID NO: 5), wherein Xaa₃ is Asn or Lys, Xaa₄ is Ala or Val, and Xaa₅ is Asn or Thr,

Formula III: Asp-Asn-Trp-Leu-Xaa₆-Tyr (SEQ ID NO: 6), wherein Xaa₆ is Ser or Thr,

Formula IV: Lys-Ser-Ser-Xaa₇-Ser-Leu-Leu-Ala-Xaa₈-Gly-Asn-Xaa₉-Xaa₁₀-Asn-Tyr-Leu-Ala (SEQ ID NO: 7), wherein Xaa₇ is H is, Arg, Gln, or Lys, Xaa₈ is Ser or Trp, Xaa₉ is H is or Gln, and Xaa₁₀ is Lys or Asn,

Formula V: Trp-Xaa₁₁-Ser-Xaa₁₂-Arg-Val-Xaa₁₃ (SEQ ID NO: 8), wherein Xaa₁₁ is Ala or Gly, Xaa₁₂ is Thr or Lys, and Xaa₁₃ is Ser or Pro, and

Formula VI: Xaa₁₄-Gln-Ser-Tyr-Ser-Xaa₁₅-Pro-Xaa₁₆-Thr (SEQ ID NO: 9), wherein Xaa₁₄ is Gly, Ala, or Gln, Xaa₁₅ is Arg, H is, Ser, Ala, Gly, or Lys, and Xaa₁₆ is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or the antigen-binding fragment may comprise

(i) a heavy variable region comprising (a) a polypeptide (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, (b) a polypeptide (CDR-H2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and (c) a polypeptide (CDR-H3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and

(ii) a light variable region comprising (a) a polypeptide (CDR-L1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, (b) a polypeptide (CDR-L2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and (c) a polypeptide (CDR-L3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected into humans for the purpose of medical treatment. Thus, chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing optimized human antibodies for accepting CDRs of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be recombinant or synthetic.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody includes a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2 (α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region V_(H) that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, C_(H1), C_(H2), and C_(H3), and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region V_(L) that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region C_(L).

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

In the anti-c-Met antibody or an antigen-binding fragment thereof, the portion of the light chain and the heavy chain excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, that is the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

The term “hinge region,” as used herein, refers to a region between CH₁ and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin may be replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid residue of the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may comprise a hinge region comprising the amino acid sequence of SEQ ID NO: 100 (U7-HC6), 101 (U6-HC7), 102 (U3-HC9), 103 (U6-HC8), or 104 (U8-HC5), or a hinge region comprising the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). Preferably, the hinge region comprises the amino acid sequence of SEQ ID NO: 100 or 101.

In one embodiment of the anti-c-Met antibody or antigen-binding fragment, the variable domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and the variable domain of the light chain comprises the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99, or 107.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 6, 2009, under Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to Korean Patent Publication No. 2011-0047698, the entire disclosure of which are incorporated herein by reference). The anti-c-Met antibody may include all the antibodies defined in Korean Patent Publication No. 2011-0047698.

In the anti-c-Met antibody, the portion of the light chain and the heavy chain excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, that is the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgG1, IgG2, and the like).

By way of further example, the anti-c-Met antibody or the antibody fragment may comprise:

(i) a heavy chain comprising an amino acid sequence selected from the group consisting of (a) the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1^(st) to 17^(th) positions is a signal peptide), (b) the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, (c) the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), (d) the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, (e) the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1^(st) to 17^(th) positions is a signal peptide), and (f) the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66; and

(ii) a light chain comprising an amino acid sequence selected from the group consisting of (a) the amino acid sequence of SEQ ID NO: 68 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), (b) the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68, (c) the amino acid sequence of SEQ ID NO: 70 (wherein the amino acid sequence from the 1^(st) to 20^(th) positions is a signal peptide), (d) the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70, and (e) the amino acid sequence of SEQ ID NO: 108.

For example, the anti-c-Met antibody may be selected from the group consisting of:

(i) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, and a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68;

(ii) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, and a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68;

(iii) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66, and a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68;

(iv) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, and a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70;

(v) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, and a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70;

(vi) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66, and a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 70;

(vii) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, and a light chain comprising the amino acid sequence of SEQ ID NO: 108;

(viii) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, and a light chain comprising the amino acid sequence of SEQ ID NO: 108; and

(ix) an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66, and a light chain comprising the amino acid sequence of SEQ ID NO: 108.

The polypeptide with the amino acid sequence of SEQ ID NO: 70 is a light chain comprising the human kappa (κ) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 of the amino acid sequence of SEQ ID NO: 70 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 of the amino acid sequence of SEQ ID NO: 70 (position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of the amino acid sequence of SEQ ID NO: 68; positioned within CDR-L1) with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibits increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

In another embodiment, the anti c-Met antibody may include a light chain complementarity determining region comprising the amino acid sequence of SEQ ID NO: 106, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107, or a light chain comprising the amino acid sequence of SEQ ID NO: 108.

The anti-c-Met/anti-EGFR bispecific antibody can not only inhibit the activity of c-Met and EGFR by the internalization and degradation activity of anti-c-Met antibody but also fundamentally block them by reducing the total amounts of c-Met and EGFR by the degradation thereof. Accordingly, the anti-c-Met/anti-EGFR bispecific antibody can obtain efficient effects even when applied to patients who have developed resistance against pre-existing anti-EGFR antibodies.

Another embodiment provides a composition for preventing and/or treating a cancer including an anti-c-Met/anti-EGFR bispecific antibody as an active ingredient.

Another embodiment provides a method of prevention and/or treatment a cancer, including administering a pharmaceutical effective amount of the anti-c-Met/anti-EGFR bispecific antibody to a patient in need of the prevention and/or treatment of the cancer. The method of prevention and/or treatment a cancer may further comprise a step of identifying the patient in need of the prevention and/or treatment of the cancer, prior to the step of administering.

Another embodiment provides a use of the anti-c-Met/anti-EGFR bispecific antibody for the prevention and/or treatment of a cancer.

The cancer may be associated with overexpression and/or abnormal activation of c-Met and/or EGFR. The cancer may be a solid cancer or hematological cancer and for instance, may be, but not limited to, one or more selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head or neck cancer, brain cancer, and the like. In particular, the cancer may be a cancer having resistance against pre-existing anticancer drugs, for example, antagonists against EGFR. The cancer may include a metastatic cancer as well as a primary cancer.

The prevention and/or treatment effects of the cancers may include effects of not only suppressing the growth of the cancer cells but also suppressing deterioration of cancers due to migration, invasion, and/or metastasis thereof. Therefore, the curable cancers may include both primary cancers and metastatic cancers. Thus, the pharmaceutical composition or method may be for preventing and/or treating cancer metastasis.

The pharmaceutically effective amount of the anti-c-Met/anti-EGFR bispecific antibody may be administered along with a pharmaceutically acceptable carrier, diluent, and/or excipient.

The pharmaceutically acceptable carrier to be included in the composition may be those commonly used for the formulation of antibodies, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and preservative.

The pharmaceutical composition or the anti-c-Met/anti-EGFR bispecific antibody may be administered orally or parenterally. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

A suitable dosage of the pharmaceutical composition or the anti-c-Met/anti-EGFR bispecific antibody may be prescribed in a variety of ways, depending on factors such as formulation methods, administration methods, age of patients, body weight, gender, pathologic conditions, diets, administration time, administration route, excretion speed, and reaction sensitivity. A desirable dosage of the pharmaceutical composition or the anti-c-Met/anti-EGFR bispecific antibody may be in the range of about 0.001 to 100 mg/kg for an adult. The term “pharmaceutically effective amount” used herein refers to an amount exhibiting effects in preventing or treating cancer.

The pharmaceutical composition or the anti-c-Met/anti-EGFR bispecific antibody may be formulated with a pharmaceutically acceptable carrier and/or excipient into a unit or a multiple dosage form by a method easily carried out by a skilled person in the pertinent art. The dosage form may be a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further include a dispersing or a stabilizing agent.

In addition, the pharmaceutical composition or the anti-c-Met/anti-EGFR bispecific antibody may be administered as an individual drug, or together with other drugs, and may be administered sequentially or simultaneously with pre-existing drugs.

Since the pharmaceutical composition includes an antibody or an antigen binding fragment thereof, it may be formulated as an immunoliposome. The liposome containing an antibody may be prepared using a well-known method in the pertinent art. The immunoliposome is a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide exchange reaction. A chemical drug such as doxorubicin may be additionally included in the liposome.

The subject to which the pharmaceutical composition is administered or the patient to which the prevention and/treatment method is applied may be mammals, for example, primates such as humans and monkeys, or rodents such as rats and mice, but are not be limited thereto. The subject or the patient may be a cancer patient having resistance against pre-existing anticancer drugs, for example, antagonists against the target cell membrane proteins.

In addition, Applicants determined that an anti-EGFR scFv fragment comprising the heavy chain variable region of the anti-EGFR antibody and the light chain variable region of the anti-EGFR antibody comprising a new amino acid sequence had excellent activity, as well as excellent stability.

Accordingly, another embodiment of the present invention provides a polypeptide comprising the amino acid sequence of SEQ ID NO: 113 or SEQ ID NO: 114. Another embodiment provides a polynucleotide encoding the polypeptide. Another embodiment provides a recombinant vector containing the polynucleotide. Another embodiment provides a recombinant cell transformed with the recombinant vector.

The term “vector” used herein refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector and an adeno-associated virus vector. Suitable recombinant vectors may be constructed by manipulating plasmids often used in the art (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, and the like), a phage (for example, λgt4λB, λ-Charon, λΔz1, M13, and the like), or a virus (for example, SV40, and the like), but not be limited thereto.

In the recombinant vector, the polynucleotides may be operatively linked to a promoter. The term “operatively linked” used herein refers to a functional linkage between a nucleotide expression regulating sequence (for example, a promoter sequence) and other nucleotide sequences. Thus, the regulating sequence may regulate the transcription and/or translation of the other nucleotide sequences by being operatively linked.

The recombinant vector may be constructed typically for either cloning or expression. The expression vector may be any ordinary vectors known in the pertinent art for expressing an exogenous protein in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

The recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when a prokaryotic cell is used as a host cell, the expression vector used generally includes a strong promoter capable of initiating transcription (for example, pL^(λ) promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, and the like), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. When a eukaryotic cell is used as a host cell, the vector used generally includes the origin of replication acting in the eukaryotic cell, for example, a f1 replication origin, a SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, or a BBV replication origin, but is not limited thereto. A promoter in an expression vector for a eukaryotic host cell may be a promoter derived from the genomes of mammalian cells (for example, a metallothionein promoter, and the like) or a promoter derived from mammalian viruses (for example, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, a tk promoter of HSV, and the like). A transcription termination sequence in an expression vector for a eukaryotic host cell may be, in general, a polyadenylation sequence.

The recombinant cell may be those obtained by transfecting the recombinant vector into a suitable host cell. Any host cells known in the pertinent art to enable stable and continuous cloning or expression of the recombinant vector may be used as the host cell. Suitable prokaryotic host cells may be one or more selected from E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus species strains such as Bacillus subtillis, or Bacillus thuringiensis, intestinal bacteria and strains such as Salmonella typhymurum, Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells to be transformed may be one or more selected from yeasts, such as Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, for example, Sp2/0, Chinese hamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines, but not be limited thereto.

The polynucleotide or the recombinant vector including the same may be transferred (transfected) into a host cell by using known transfer methods. Suitable transfer methods for prokaryotic host cells may include a method using CaCl₂ and electroporation. Suitable transfer methods for eukaryotic host cells may include microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment, but are not limited thereto.

A transformed host cell may be selected using a phenotype expressed by a selected marker by any methods known in the art. For example, if the selected marker is a gene that is resistant to a specific antibiotic, a transformant may be easily selected by being cultured in a medium including the antibiotic.

Another embodiment provides a heavy chain variable region of the anti-EGFR antibody comprising the amino acid sequence of SEQ ID NO: 113. Another embodiment provides a light chain variable region of the anti-EGFR antibody comprising the amino acid sequence of SEQ ID NO: 114. Another embodiment provides an anti-EGFR scFv fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 113 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 114. The anti-EGFR scFv fragment can maintain its binding activity to EGFR for 120 hours or more, 140 hours or more, or 160 hours or more and thus, has significantly high antibody stabilities.

The anti-c-Met/anti-EGFR bispecific antibody of the present invention can expect the following effects, compared to the existing anti c-Met antibodies:

1. Cancer cell inhibitory effects increased more than the existing anti-c-Met antibodies or anti-EGFR drugs.

2. Inhibitory effects in cancer cells having resistance to the existing anti-c-Met antibodies or anti-EGFR drugs.

3. Effects of stability increase of bispecific antibody through the engineering of anti-EGFR scFv.

Hereafter, the present invention will be described in detail by examples.

The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

EXAMPLES Reference Example 1 Construction of Anti-c-Met Antibody

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×10⁸ cells) were mixed with myeloma cells (Sp2/0) (1×10⁸ cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×10⁵ cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO₂ incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 9, 2009, under Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO₂ incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with a filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Reference Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL, and after 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in an 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO₂ condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO₂ condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (IgBLAST online database tool, maintained by National Center for Biotechnology Information (NCBI), Bethesda, Md.) search revealed that VH3-71 has an identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search revealed that VK4-1 has an identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a BLAST search. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have an identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A BLAST search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL, and after 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker having the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 1, below.

TABLE 1 CDR Amino Acid Sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 2 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.

TABLE 2 Library  Clone constructed CDR Sequence H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37) 1.7. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL, and after 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DN:light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain including the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain including a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain including the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a vector from the pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a vector from the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL, and after 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (U6-HC7) and huAbF46-H4-A1 (IgG2 Fc) were selected for the following examples, and respectively referred as anti-c-Met antibody L3-1Y U6-HC7 for huAbF46-H4-A1(U6-HC7) and anti-c-Met antibody L3-1Y IgG2 for huAbF46-H4-A1(IgG2 Fc).

Reference Example 2 Preparation of Anti-EGFR scFv

The sequence of an scFv of the antibody binding to EGFR was prepared on the basis of the sequence of Cetuximab, which is a chimeric antibody, or on the basis of the sequence of a rat-human chimeric antibody by inserting a (GGGGS)₃ (SEQ ID NO: 115) linker peptide between the heavy chain variable region and the light chain variable region. In particular, the DNA sequence encoding a (GGGGS)₃ (SEQ ID NO: 115) linker peptide was added to the DNA sequence (SEQ ID NO: 110) encoding the heavy chain variable region (SEQ ID NO: 109) and the DNA sequence (SEQ ID NO: 112) encoding the light chain variable region (SEQ ID NO: 111) of a humanized anti-EGFR antibody using an automatic gene synthesis (Bioneer Inc.) to synthesize a DNA fragment encoding an scFv of the anti-EGFR antibody.

A modified anti-EGFR scFv (heavy chain variable region: SEQ ID NO: 113; and light chain variable region: SEQ ID NO: 114) was prepared as described above, with the exception that the amino acid, F, at 51^(st) position of the heavy chain variable region (SEQ ID NO: 109) was substituted with I, the amino acid G at 44^(th) position with C, and the amino acid Q at 62^(nd) position with S, and the amino acid R at 46^(th) position of the light chain variable region (SEQ ID NO: 111) was substituted with L, the amino acid F at 83^(rd) position with E, and the G at 100^(th) position with C. The amino acid location within the antibody complies with kabat numbering system.

The thus obtained anti-EGFR scFv or modified anti-EGFR scFv was used to manufacture the following bispecific antibodies.

Example 1 Preparation of Bispecific Anti-c-Met/Anti-EGFR Antibodies

The anti-EGFR scFv or modified anti-EGFR scFv prepared in Reference Example 2 was fused at the C-terminus of the Fc of the anti c-Met antibody L3-1Y-IgG2 (huAbF46-H4-A1 (IgG2 Fc)) prepared in Reference Example 1. The fusion procedures are as follows.

A DNA segment having a base sequence (SEQ ID NO: 66) corresponding to the heavy chain of the anti c-Met antibody L3-1Y-IgG2 prepared in Reference Example 1 was inserted into a vector of the pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) which is included in OptiCHO™ Antibody Express Kit (Cat no. 12762-019) by Invitrogen Inc., and a DNA segment having a base sequence (SEQ ID NO: 68) corresponding to the light chain of the anti c-Met antibody L3-1Y-IgG2 was inserted into a vector of the pOptiVEC™-TOPO TA Cloning Kit. Thereafter, the anti-EGFR scFv encoding DNA prepared in Reference Example 2 was fused at the C-terminus of the Fc of L3-1Y-IgG2 inserted into pcDNA™3.3, using the coding DNA sequence of a linker peptide having 10 amino acid lengths consisting of (G₄S)₂ (SEQ ID NO: 116), to construct vectors for the expression of bispecific antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (Invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×10⁵ cells/mL, and after 24 hours, when the cell number reached to 1×10⁶ cells/mL, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (Invitrogen), wherein in a 15 mL tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA: light chain DNA) and mixed with 2 mL of OptiPro™ SFM (Invitrogen) (A). In another 15 mL tube, 100 μL of Freestyle™ MAX reagent and 2 mL of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO₂.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE Healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was replaced by a PBS buffer to finally obtain purified bispecific anti c-Met/anti-EGFR antibodies.

The thus prepared anti-c-Met/anti-EGFR bispecific antibody in which the anti-EGFR scFv is fused at the C-terminus of the anti-c-Met antibody L3-1Y-IgG2 was named ME-01, and the anti-c-Met/anti-EGFR bispecific antibody in which the modified anti-EGFR scFv is fused at the C-terminus of L3-1Y-IgG2 was named ME-03S.

Example 2 Dual Binding of Anti-c-Met/Anti-EGFR Bispecific Antibody

Whether the anti-c-Met/anti-EGFR bispecific antibody is an antibody against two types of antigens (c-Met/EGFR) was identified using Biacore T100 (GE). An anti-6×His antibody (#MAB050, R&D Systems) was immobilized onto a CM5 chip (#BR-1005-30, GE) using an amine coupling kit (#BR-1000-50, GE) according to the manufacturer's instructions. After the immobilization, c-Met-Fc (#358-MT/CF, R&D Systems) was injected thereto at a concentration of 15 μg/mL for 1 min and, then, the bispecific antibody ME-03S prepared in Example 1 was injected at a concentration of 20 nM for 1 min. Thereafter, EGFR-Fc (#344-ER, R&D Systems) was injected at 100 nM for 1 min and, then, association/dissociation was observed.

The obtained results are shown in FIG. 2. As seen in FIG. 2, the bispecific antibody ME-03S prepared in Example 1 bound to both c-Met and EGFR.

In addition, the affinities of the prepared anti-c-Met/anti-EGFR bispecific antibody toward two types of antigens (c-Met/EGFR) were each identified using Biacore T100 (GE). A human Fab binder (GE Healthcare) was immobilized at the surface of a CM5 chip (#BR-1005-30, GE) according to the manufacturer's specifications. About 90 to 120 Ru of ME-03S was captured, and c-Met-Fc (#358-MT/CF, R&D Systems) or EGFR-Fc (#344-ER, R&D Systems) were injected at various concentrations into the captured antibody. 10 mM Glycine-HCl (pH 1.5) solution was injected thereto to regenerate the surface. In order to measure affinity, the data obtained from this experiment was fitted using BIAevaluation software (GE Healthcare, Biacore T100 evaluation software).

The obtained results are shown in Table 3 below.

TABLE 3 Antibody Antigen K_(D) (nM) k_(a) (1/Ms) k_(d) (1/s) ME03S cMet 0.14 6.6 × 10⁵ 9.3 × 10⁻⁵ EGFR 0.12 2.4 × 10⁵ 2.7 × 10⁻⁵

As seen in Table 3, the bispecific antibody ME03S prepared in the Example 1 bound to both c-Met and EGFR.

Reference Example 3 Preparation of Cell Lines

Cell lines to be used in the following examples for the efficiency test of the anti-c-Met/anti-EGFR bispecific antibody prepared in Example 1 are EBC1, a lung cancer cell line, MKN45 and HCC827, stomach cancer cell lines, and HCC827 ER #10 and HCC827 ER #15 which are resistance microbes having resistance against Erlotinib (Er) which is an inhibitor against EGFR in the HCC827 cell line.

The EBC1 lung cancer cell line and the MKN45 stomach cancer cell line were purchased from ATCC. The HCC827 stomach cancer cell line, and the resistance microbes HCC827 ER #10 and HCC827 ER #15, were obtained from Samsung Medical Center, Korea.

Example 3 Cancer Cell Proliferation Inhibition Test of Anti-c-Met/Anti-EGFR Bispecific Antibody

Suppression effects on the proliferation of cancer cells of the anti-c-Met/anti-EGFR bispecific antibody prepared in Example 1 were identified in the MKN45 stomach cancer cell line.

The cell lines were cultured in a RPMI1640 medium (#11875-093, Gibco) to which 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin were added, in 5% CO₂ and 37° C. conditions. For cell proliferation assay, each cell line was subcultured at a concentration of 1×10⁴ cells/well in a 96-well plate, which was treated with the anti-c-Met/anti-EGFR bispecific antibody ME-03S prepared in Example 1 in an amount of 5 μg/mL and cultured for 72 hours. A medium with no antibody added was used as a negative control, and commercially available EGFR inhibitors Erlotinib (marked as Er; #S1023, Selleckchem; 2 μM), Erbitux (marked as Ebt; #ET509081213, Merck; 5 μg/mL), L3-1Y-IgG2 antibody (5 μg/mL) prepared in Reference Example 1, and co-treatment group of L3-1Y antibody prepared in Reference Example 1 and Erbitux were each used as positive controls.

After cultivation, cell proliferation degrees were analyzed using Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, Md.) according to the manufacturer's instructions. In brief, after the cultivation for 72 hours, 10 μL of CCK8 solution was added to each well and after the additional cultivation for 2.5 hours, absorption degrees were read at 450 nm using a microplate reader.

The obtained results are shown in FIG. 3. As seen in FIG. 3, the anti-c-Met/anti-EGFR bispecific antibody ME-03S showed remarkable increases in cell proliferation inhibitory effects as compared to the cases treated individually with the anti-c-Met antibody L3-1Y and the anti-EGFR antibody Erbitux (Er). In particular, the fusion of the anti-c-Met antibody L3-1Y and the anti-EGFR antibody Erbitux showed excellent cell proliferation inhibitory effects, even compared to the co-treatment case (L+E), which is not a form of bispecific antibody.

Example 4 c-Met and EGFR Activation Inhibitory Effect of Anti-c-Met/Anti-EGFR Bispecific Antibody

The MKN45 cell line and the HCC827 cell line (HCC827 WT, HCC827 #15) were each treated with Gefitinib (#S1025, Selleckchem), Erlotinib, Erbitux, L3-1Y-IgG2 antibody (Reference Example 1), and ME-03S (Example 1), individually or in combination for 24 hours. Specific treatment in each cell is as follows:

MKN45 cell line: Erlotinib 10 μg/mL, Erbitux 5 μg/mL, L3-1Y 5 μg/mL, ME-03S 5 μg/mL, (1% (v/v) DMSO for 24 hrs with or without EGF 100 ng/mL for 30 min)

HCC827: Gefitinib 1 μM, Erlotinib 2 μM, L3-1Y 5 μg/mL, ME03S 5 μg/mL, (1% (v/v) DMSO for 24 hrs with or without EFG 50 ng/mL).

Thereafter, the cells were lysed with Complete Lysis-M (#04719956001, Roche) to collect cell lysates, which were then subject to SDS-PAGE electrophoresis.

After the electrophoresis, the proteins from the gels were transferred onto a nitrocellulose membrane (#LC2006, Invitrogen), which was then blocked with a skim milk (5% in TBST) at a room temperature for one hour. After blocking, for the primary antibody reaction, the membrane was treated with a 1:1000 dilution of anti-phospho c-Met antibody, anti-phospho EGFR antibody, anti-EGFR antibody (all available from Cell signaling technology), and anti-c-Met antibody (Abcam) in an amount of 10 μg/mL at a room temperature for two hours. After the primary antibody reaction, the membrane was washed three times with PBS for 5 min and, then, it was treated with a horseradish peroxidase attached secondary antibody (Cell signaling technology) against each primary antibody in an amount of 1 μg/mL at a room temperature for one hour. After the secondary antibody reaction, the membrane was washed three times with PBS for 5 min and then sensitized with Enhanced chemiluminescence substrate (Thermo scientific) to measure the degree of antigen-antibody reaction through ImageQuant LAS system (GE Healthcare). The EGF was treated at a concentration of 100 ng/mL before 30 min. of cell lysate formation.

The obtained results are shown in FIGS. 4 to 6. As seen in FIGS. 4 to 6, when treated with the anti-c-Met/anti-EGFR bispecific antibody ME-03S, it showed excellent c-Met phosphorylation suppression and c-Met degradation degree, compared to the sole treatment of the anti-c-Met antibody L3-1Y and the co-treatment of the L3-1Y and EGFR inhibitor erlotinib. Also, in the sole treatment of the EGFR inhibitor and the co-treatment of the L3-1Y and EGFR inhibitor erlotinib, the phosphorylation suppression of EGFR was observed but EGFR was hardly degraded. However, in the treatment of the anti-c-Met/anti-EGFR bispecific antibody ME-03S, not only the phosphorylation suppression of EGFR but also the degradation of EGFR was observed. This is to show that the anti-c-Met/anti-EGFR bispecific antibody ME-03S has not only the phosphorylation inhibitory activity of EGFR but also the degradation activity of EGFR, and to show that the anti-EGFR antibody has synergistic effects by forming a bispecific antibody together with the anti-c-Met antibody, compared to the sole EGFR inhibitor.

Example 5 Co-Localization of c-Met and EGFR by Anti-c-Met/Anti-EGFR Bispecific Antibody

The MKN45 cell line and the EBC1 cell line were prepared in amounts of 4×10⁴ cell/well, respectively, to which L3-1Y-IgG2 prepared in Reference Example 1, anti-EGFR scFv prepared in Reference Example 2, and the anti-c-Met/anti-EGFR bispecific antibody ME03S prepared in Example 1 were each added individually or in combination to result in the concentration of 1 μg/mL per well (in case of the combination treatment, to result in 1 μg/mL each) and treated at 37° C. for 4 hours. The cells were treated with 4% (v/v) formaldehyde for 15 min to immobilize them on a plate, and washed three times with PBS. Thereafter, the cells were treated with a blocking buffer (0.5% triton x-100 and 5% donkey serum) at a room temperature for one hour and then treated with a 1:100 dilution of the first antibodies (c-Met primary antibody; #FAB3582A, R&D systems, EGFR primary antibody; #5616, Cell signaling) against c-Met and EGFR, respectively in amounts of 100 μA at 4° C. for 15 hours. After the cells were washed three time with PBS, they were treated with a 1:2000 dilution of the secondary antibody (#A21433, Invitrogen) in an amount of 100 μA at a room temperature for 1 hour and washed three times with PBS to prepare a plate with a mounting medium (#H-1200, Vector). The prepared cells were observed with a confocal microscope (Zeiss, LSM710).

The obtained results are shown in FIG. 7 (MKN45 stomach cancer cell line) and FIG. 8 (EBC-1 lung cancer cell line). As seen in FIG. 7 and FIG. 8, in the case of the sole treatment of anti-EGFR scFv, EGFR still locates on cell membranes. Even in the case of the co-treatment of L3-1Y and anti-EGFR scFv, only c-Met migrates to inside of the cells and EGFR still locates at the cell membranes; however, in the case of the treatment of the anti-c-Met/anti-EGFR bispecific antibody ME03S, both c-Met and EGFR migrated inside the cells.

Example 6 Stability of Bispecific Anti-c-Met/Anti-EGFR Antibodies

The bispecific anti c-Met/anti-EGFR antibodies ME-03 and ME-03S prepared in the Example 1 were mixed with rat serum to obtain samples having the concentration of 125 ng/mL, which were then reacted at 37° C. After the lapse of the designated time (see FIG. 9), each sample was stored in a deep freezer.

An ELISA was performed to determine the binding of the bispecific anti c-Met/anti-EGFR antibodies mixed in serum to antigens over time lapse. In particular, c-Met and EGFR were immobilized at the concentration of 2 μg/mL on a microplate and then reacted with blocking solution (5% milk, 0.1% Tween 20) at a room temperature for 30 min. Thereafter, 100 μl of a 1:3000 dilution of the prepared samples was reacted with the antigens (c-Met and EGFR) immobilized on the plate at a room temperature for one hour and washed. After that, they were treated with goat anti-human Fab-HRP (#15260, Sigma-Aldrich) and then washed. They were then treated with TMB as a substrate for color development and their absorption was measured at 450 nm.

The obtained results are shown in FIG. 9. As seen in FIG. 9, the bispecific anti c-Met/anti-EGFR antibodies maintained binding activity to their antigens in a stable manner for relatively long time. Especially, the bispecific anti c-Met/anti-EGFR antibody ME-03S had superior stability by maintaining binding activity to its antigen for at least 168 hours. This result demonstrates that a sequence mutation within the frame of anti-EGFR scFv as in Reference Example 2 can increase the stability of the antibodies.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. 

What is claimed is:
 1. An anti-c-Met/anti-EGFR bispecific antibody comprising (a) an anti-c-Met antibody or an antigen-binding fragment thereof and (b) an anti-EGFR antibody or an antigen-binding fragment thereof, wherein the anti-c-Met antibody or the antigen-binding fragment thereof comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:
 37. 2. The anti-c-Met/anti-EGFR bispecific antibody according to claim 1, wherein the antigen-binding fragment is scFv, (scFv)2, scFvFc, Fab, Fab′ or F(ab′)₂.
 3. The anti-c-Met/anti-EGFR bispecific antibody according to claim 1, wherein the anti-c-Met antibody or the antigen-binding fragment thereof comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 62, the amino acid sequence from the 18^(th) to 462^(nd) positions of the amino acid sequence of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64, the amino acid sequence from the 18^(th) to 461^(st) positions of the amino acid sequence of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66, or the amino acid sequence from the 18^(th) to 460^(th) positions of the amino acid sequence of SEQ ID NO: 66; and (ii) a light chain comprising the amino acid sequence of SEQ ID NO: 68, the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70, or the amino acid sequence from the 21^(st) to 240^(th) positions of the amino acid sequence of SEQ ID NO:
 70. 4. The anti-c-Met/anti-EGFR bispecific antibody according to claim 1, wherein the antigen-binding fragment of the anti-EGFR antibody is: (a) an antigen binding fragment of Cetuximab, (b) an antigen binding fragment of Panitumumab, or (c) an antigen binding fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 113, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO:
 114. 5. The anti-c-Met/anti-EGFR bispecific antibody according to claim 1, comprising an anti-c-Met antibody and an antigen-binding fragment of an anti-EGFR antibody, wherein the antigen-binding fragment of the anti-EGFR antibody is coupled to the C terminus of the anti-c-Met antibody.
 6. A method of treatment of a cancer, comprising administering the anti-c-Met/anti-EGFR bispecific antibody of claim 1 to a patient in need thereof.
 7. The method according to claim 6, wherein the antigen-binding fragment is scFv, (scFv)2, scFvFc, Fab, Fab′ or F(ab′)₂ of the antibody.
 8. The method according to claim 6, wherein the antigen-binding fragment of the anti-EGFR antibody is (a) an antigen binding fragment of Cetuximab, (b) an antigen binding fragment of Panitumumab, or (c) an antigen binding fragment comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 113, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO:
 114. 9. The method according to claim 6, wherein the anti-c-Met/anti-EGFR bispecific antibody comprises an anti-c-Met antibody and an antigen-binding fragment of an anti-EGFR antibody, wherein the antigen-binding fragment of the anti-EGFR antibody is coupled to the C terminus of the anti-c-Met antibody. 